Clathrin light chain‐conjugated drug delivery for cancer

Abstract Targeted drug delivery systems hold the remarkable potential to improve the therapeutic index of anticancer medications markedly. Here, we report a targeted delivery platform for cancer treatment using clathrin light chain (CLC)‐conjugated drugs. We conjugated CLC to paclitaxel (PTX) through a glutaric anhydride at high efficiency. Labeled CLCs localized to 4T1 tumors implanted in mice, and conjugation of PTX to CLC enhanced its delivery to these tumors. Treatment of three different mouse models of cancer—melanoma, breast cancer, and lung cancer—with CLC‐PTX resulted in significant growth inhibition of both the primary tumor and metastatic lesions, as compared to treatment with free PTX. CLC‐PTX treatment caused a marked increase in apoptosis of tumor cells and reduction of tumor angiogenesis. Our data suggested HSP70 as a binding partner for CLC. Our study demonstrates that CLC‐based drug‐conjugates constitute a novel drug delivery platform that can augment the effects of chemotherapeutics in treating a variety of cancers. Moreover, conjugation of therapeutics with CLC may be used as means by which drugs are delivered specifically to primary tumors and metastatic lesions, thereby prolonging the survival of cancer patients.


| INTRODUCTION
Targeted drug delivery systems can amplify the concentration of transported payloads at various tissues of interest, including tumors. [1][2][3] Thus, a key advantage of targeted drug delivery is its simultaneous enhancement of the therapeutic index of the drug, along with a reduction in its systemic exposure and overall toxicity. 3 Antibody-drug conjugates (ADCs) are emerging platforms for the delivery of a range of cancer drugs. 4,5 Monoclonal antibodies have attracted major interest as vehicles for the delivery of cancer therapeutics by recognizing specific target antigens overexpressed on the surface of cancer cells. The molecular weight of antibodies affects the tumor penetration of ADCs, and the large size of IgG antibodies (~150 kDa) used widely for current ADCs presents a notable challenge. 6 The immunogenicity of antibodies also results in systematic toxicity and their rapid clearance from the body, culminating in the delivery of a small fraction of the administered drug. 7 Therefore, efforts in identifying alternative approaches to achieve targeted therapy have heightened. One such route is the use of a small protein for disease treatment. [8][9][10] Advantages to the use of small proteins include easy and affordable production, high pharmaceutical potency and flexibility, as well as low toxicity in sequence and conjugation possibilities.
Here, we report a novel application of clathrin light chain A (CLC)-based drug delivery. CLC is an endogenous small protein that forms a network of triskelions that constitute a polyhedral lattice around vesicles that assist in the sorting of cargo for intracellular trafficking. 11 Clathrin-coated vesicles play an essential role in cellular membrane trafficking. The clathrin triskelion consists of three heavy chains and three light chains. 11 During clathrin-mediated endocytosis, this cytosolic clathrin triskelion interacts with other cytosolic proteins, including HSP70, and regulates the formation of clahtrin-coated vesicles. 12 The light chain is a primary functional unit of this triskelion via its interaction with calcium ions 13 or calmodulin, 14 or through its phosphorylation. 15 Endogenous proteins, such as ferritin and albumin, have attracted great interest in the field of drug delivery, due to their biocompatibility and favorable safety profiles. [16][17][18][19][20][21][22] In the same vein, CLC piqued our interest as a novel endogenous small protein that could be used as a carrier of payloads.
First, we confirmed that CLCs interact with cancer cells and could be used as vehicles for targeted delivery to malignant tumors. Next, CLCs were conjugated to the antineoplastic agent paclitaxel (PTX) via reaction with glutaric anhydride (CLC-PTX) to evaluate their utility for cancer-targeted delivery and treatment in a series of mouse models.
CLC-PTX enhanced the concentration of PTX at the tumor site and suppressed tumor growth in mouse models of breast cancer, melanoma, and lung carcinoma. In addition, CLC-PTX was found to reduce the size of metastatic lesions in breast cancer.

| RESULTS
2.1 | We synthesized and characterized CLC-PTX conjugates CLC-6 histone (MW~28 kDa) was expressed in Escherichia coli (E. coli), confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and extracted at a purity of >85%, as demonstrated by Western blot (data not shown). PTX is a chemotherapeutic drug used for the treatment of numerous cancers. [23][24][25][26][27][28] The use of PTX has several drawbacks, such as poor solubility and tissue toxicity 23 that render it a prime candidate for drug-carrier conjugation.
Therefore, we used a pH-sensitive linker to conjugate CLC to PTX ( Figure S1). We conjugated CLC with PTX via esterification of C-2 0 in PTX by glutaric anhydride, a step that removes the cytotoxicity of PTX. 29 We confirmed the synthesis of 2'-glutaryl PTX by 1  Commercially available fluorescent PTX (Oregon Orange-tagged PTX, abbreviated as PTX*) was used to confirm the conjugation of PTX to CLC. The maximal absorbance of PTX* is observed at a wavelength of around 500 nm. We conjugated 2 0 -glutaryl PTX* to CLC via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/sulfo-N-hydroxysuccinimide (NHS) coupling to produce CLC-PTX*, which was verified by ultraviolet-visible (UV-Vis) spectroscopy ( Figure 1c).
The absorbance of CLC-PTX* was compared to unconjugated CLC, revealing absorption in the visible wavelength region, originating from PTX*. The ratio of PTX* to CLC was 1.1 ± 0.2, based on the extinction coefficients (ε) of CLC (λ = 280 nm) and PTX* (λ = 500 nm). The drug to antibody ratio (DAR) of PTX to CLC was confirmed again as 1.0 ± 0.1 by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), a similar value as above (Figure 1d). After conjugation of PTX, the hydrodynamic size of CLC in phosphate-buffered saline (PBS) increased from 6.6 ± 2.6 to 10.9 ± 2.2 nm (Figure 1e), whereas the zeta-potential of CLC changed from À4.0 ± 2.6 mV to À25.7 ± 11.3 mV, presumably by the formation of an amide bond from a primary amine on CLC ( Figure 1f). Next, CLC-PTX* conjugates released PTX* at a higher rate over time in pH 5.0 acetate buffer in comparison to pH 7.4 phosphate buffer at 37 C (Figure 1g). Intravenous (iv) administration of CLC twice per week for 2 weeks to mice resulted in no notable toxicity to the lungs, liver, kidneys, or heart, as determined by histological observation ( Figure S2A). In addition, no significant increase in serum creatinine and blood urea nitrogen (BUN), serologic markers of kidney injury, was noted in mice following treatment with CLC ( Figure S2B and C). siRNA. (f) CLC signal interacted with 4T1 cells with or without knockdown of HSP70 by siRNA. Significance was determined by Student's t-test. The data are represented by means ± SD (**p < 0.01)

| Depletion of HSP70 in 4T1 cells hinders the uptake of CLCs
We sought to determine whether CLC was internalized by 4T1 mouse breast cancer cells by incubating these cells with CLCs attached to Alexa Fluor™ 488 dye. The colocalization of the CLCs with a lysosome marker was confirmed (Figure 2a), which indicated that the CLCs underwent endocytosis and were shuttled to acidic lysosomes in the 4T1 cells.
A virtual screening between CLC and a protein data bank (PDB) was conducted, resulting in the identification of three proteins with high binding energy (Table S1). Interestingly, one of these putative binding partners was HSP70, commonly reported as a cancer marker.
A number of studies indicate the expression of HSP70 on the surface of cancer cells. [30][31][32][33][34][35] We compared the interaction with CLC in 4T1 cells and HK02 kidney tubular epithelial cells. 4T1 cells interacted with CLC 11.7-fold more than HK02 cells (Figure 2b (Figure 2f), suggesting that HSP70 plays a role in the uptake of CLC by 4T1 cells. We used bovine serum albumin (BSA) as a control to rule out the possibility of nonspecific uptake of CLC by 4T1 cells.
We found that 4T1 cells internalized CLC-Alexa 594 significantly more robustly in vitro than BSA-Alexa 594 ( Figure S5D).

| CLCs localize to 4T1 breast primary tumor and metastatic lesions in mice
We examined the capacity of CLCs to localize to 4T1 breast cancer in vivo by administering CLC-IR800 (4 mg/kg) iv to 4T1 tumor-bearing BALB/c mice. The mice were euthanized at 1 day (1 d), 2 days, and 3 days following administration of CLCs, at which time points we measured the fluorescent signal of CLC-IR800 in various organs, using an iBox Explorer 2 Imaging Microscope. CLC-IR800 signal in the tumor remained stable from 1 day to 3 days, but CLC signals in other organs faded out by 3 days (Figure 3a). The signals from lung and spleen were trivial for 3 days: CLC in these organs were also cleaned out (data not shown). We administrated CLC-IR800 (4 mg/kg) to 4T1 tumorbearing BALB/c mice. After 6 h, we harvested the 4T1 tumors and stained them for HSP70. We confirmed a high overlap between the CLC and HSP70 signals ( Figure 3b). We also conjugated CLC with PTX*, then injected 4T1 bearing mice iv with CLC-PTX* and quantified the fluorescence of PTX*. PTX* did not clear significantly from the tumor within the span of the study, F I G U R E 4 Inhibition of cancer growth in 4T1 murine breast cancer model by clathrin light chain-paclitaxel (CLC-PTX). (a) Growth curves of 4T1 tumor after implantation in mice and treatment with free PTX, CLC-PTX, or PBS. Treatment was started on d 13, and drugs were administered twice per week for 2 weeks (n = 6/group). At Day 27, all the mice were euthanized because the diameter of the tumors in the PBStreated mice reached~2 cm. Arrows: treatment days. The data are represented by means ± SD (****p < 0.0001, the significance was determined by two-way ANOVA with Holm-Sidak's post hoc. (b) Fluorescence micrographs of tumors at the end of treatment with PBS (first column), free PTX (second column), and CLC-PTX (third column). Tumor sections were stained with antibodies to pancytokeratin (first row), caspase 3 (second row), ki67 (third row), CD31 (fourth row), and fibronectin (fifth row). The fluorescence intensities from the fluorescence micrographs were compared in histograms (fourth column). Scale bar: 100 μm. The data were tested by oneway ANOVA with Holm-Sidak's post hoc. The data are represented by means ± SD (n = 3, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) whereas the PTX* signal declined in the liver and the kidney over time ( Figure S6).
The lung is the most common site of metastasis for breast cancer.
Lung with 4T1 metastasis showed enhanced targeting signals of CLC as compared with naïve lung (Figure 3c). Interestingly, we observed that CLC-IR800 localized more robustly to metastatic nodules of the lung (Figure 3c, inset), supporting the concept that CLC can deliver drugs specifically to metastatic lung lesions.
We further tested whether CLC-PTX* localized to 4T1 breast cancer in vivo following iv administration to 4T1 breast tumorbearing BALB/c mice. Ex vivo fluorescent images of the tumors were acquired 1 day following administration of PTX* or CLC-PTX* at an equivalent PTX* dose of 0.5 mg/kg, which showed that the accumulation of CLC-PTX* in the tumor was higher than free PTX* ( Figure 3D). Finally, either PTX or CLC-PTX was injected iv into BALB/c mice.
At 6 h and 2 d, the sera of these mice were collected, and high-performance liquid chromatography (HPLC) quantified PTX. We detected significant amounts of PTX in the sera of the CLC-PTX group as compared with the free PTX group at 6 h, indicating that conjugation to CLC enhanced the blood circulation of PTX ( Figure 3e). The PK of CLC-PTX* derived from the fluorescence signal also revealed that conjugation with CLC prolonged the circulation of PTX ( Figure S7).

| CLC-based delivery of PTX enhances its treatment efficacy for 4T1 breast cancer
Therapeutic effects of CLC-PTX conjugates were studied in BALB/c mice bearing 4T1 breast cancer, which is refractory to antineoplastic F I G U R E 5 Inhibition of metastasis of 4T1 cancer to TDLN and lung by clathrin light chain-paclitaxel (CLC-PTX). (a) Fluorescence micrographs for the tumor-draining lymph node (TDLN) at the end of treatment (27 days postimplantation) with PBS (first column), free PTX (second column), and CLC-PTX (third column). TDLN sections were stained with antibodies to pan-cytokeratin (first row), LYVE1 (second row), and fibronectin (third row). Scale bar: 100 μm. (b) Representative photographs of the lung in each treatment group. Circle: Foci. Right graph; Number of metastatic foci per lung lobe. The data represent mean ± SD. All data were tested by one-way ANOVA with Holm-Sidak's post hoc or Student ttest (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) agents. 36 2.5 | CLC-PTX therapy disrupts metastasis of 4T1 breast cancer to the tumor-draining lymph node and lung We examined the TDLNs (inguinal lymph nodes) of 4T1 tumorbearing mice at 27 days following implantation ( Figure 5). The expression of pan-cytokeratin in the TDLN was significantly reduced by treatment with CLC-PTX, as compared to the groups that received F I G U R E 6 Inhibition of cancer growth in B16 murine melanoma model by clathrin light chain-paclitaxel (CLC-PTX). (a) Growth curves of B16 tumor after implantation in mice and treatment with free PTX, CLC-PTX, or PBS. Treatment was started on 12 days postimplantation, and drugs were administered twice per week for 2 weeks (n = 6/group). Arrows: treatment days. The data are represented by mean ± SD (**p < 0.01 and ****p < 0.0001, the significance was determined by two-way ANOVA with Holm-Sidak's post hoc. (b-e) Percentage of positive cells of melan-A (b), caspase-3 (c), Ki67 (d), and CD31 (e) analyzed in immunofluorescence staining of B16 tumor tissues. The data were tested by one-way ANOVA with Holm-Sidak's post hoc. The data are represented by means ± SD (n = 3, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) F I G U R E 7 Inhibition of cancer growth in LLC1 murine lung carcinoma model by clathrin light chain-paclitaxel (CLC-PTX). (a) Growth curves of LLC1 tumor after implantation in mice and treatment with free PTX, CLC-PTX, or PBS. Treatment was started on 12 days postimplantation of tumor, and drugs were administered twice per week for 2 weeks (n = 6/group). Arrows: treatment days. The data are represented by mean ± SD (****p < 0.0001, the significance was determined by two-way ANOVA with Holm-Sidak's post hoc. (b-d) Rate of cells stained with antibodies to pancytokeratin (b), caspase-3 (c), Ki67 (d), and CD31 (d). The data were tested by one-way ANOVA with Holm-Sidak's post hoc. The data are represented by means ± SD (n = 3, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) either PTX alone or no treatment (first row in Figure 5a). A similar decrease was also noted in the expression of LYVE-1 + lymphatic vessels and fibronectin in the TDLNs of the CLC-PTX-treated group in comparison to other groups (second and third rows in Figure 5a). We also counted the number of visible metastatic nodules of lungs of treated groups that showed no pulmonary nodules in the CLC-PTX group unlike other groups (Figure 5b).

| CLC-PTX therapy boosts growth inhibition of B16 melanoma
We also investigated the antineoplastic efficacy of CLC-PTX in a B16 melanoma mouse model, using C57BL/6 mice as the host. Treatments were administered after the tumor size reached~100 mm 3

| DISCUSSION
Targeted drug delivery systems have attracted major interest and already entered into clinical practice for various diseases, but their application to cancer remains to be developed. [38][39][40] Although chemical linkers in ADCs can be noncleavable, the majority of ADCs in clinical development have specific release mechanisms that permit the controlled release of the drugs at target sites. 41 For example, acidcleavable linkers harness the acidity within lysosomes (pH 4.5-5.0). This strategy has already yielded clinical success, as demonstrated by the products Mylotarg ®42 and Besponsa ® . 43 Esterification by anhydrides is a well-established method for drug release in the field of medicine. 44,45 Here, we used glutaric anhydride to add hydrolytically cleavable functions to CLC-based conjugates. Our conjugation method capitalizes on the obligate localization of CLC-PTX to the lysosomes as the basis for the release of PTX from CLC, activating its therapeutic function. Moreover, the small size of ADCs is critical for optimal tumor penetration. Currently, antibody fragments have been engineered, such as nanobodies, diabodies, and single-domain antibodies, to overcome the limitations of using large IgG antibodies (~150 kDa). 6,46,47 The small size of CLC can confer advantages with respect to tumor penetration in the absence of fractionation. Since CLC is a protein that is widely distributed in the human body, 48 it may have a lower toxicity profile. In addition, large-scale production of CLC using an expression system increases its utiltiy. 49 Metastasis is the primary factor of cancer morbidity and mortality. [50][51][52] Metastatic breast cancer presents a major clinical challenge, causing many deaths on a yearly basis. 53 Beyond current antibody-based treatments, 54,55 the development of other nonantibody modalities for targeted therapy is required to improve breast cancer treatment. A previous preclinical approach to target HSP70 expressed by cancer cells for in vivo tumor imaging has yielded success. 56 Vesicles coated with clathrin triskelia are critical for membrane trafficking in cells. A clathrin triskelion is composed of three heavy chains and three light chains. 11 Notably, the light chain acts as a regulatory unit in this triskelion through its binding with calcium ion 13 or calmodulin, 14 as well as its phosphorylation. 15 Importantly, HSP70 participates in one such regulatory mechanism, as it interacts directly with CLC to disassemble clathrin-coated vesicles. HSP70 targets a specific region of CLC, which is known as the "HSP70 interaction sequence," identified by anti-peptide antibodies. 57 Other studies have reported high expression of HSP70 in breast cancer, 58 melanoma, 33 and lung carcinoma. 56 HSP70 is expressed generally on the plasma membranes of primary tumor cells and distant metastases. 30 This membrane-bound HSP70 has been identified in a variety of different primary cancers. 31,32 Moreover, the density of membrane-bound HSP70 in metastatic lesions is higher than the corresponding primary tumors in mouse and human cancer models. [33][34][35] Expression of HSP70 increases further in high-grade cancers, correlating with enhanced motility, invasion, and metastasis. 59 Nonetheless, future in vivo targeting studies are required to define the mechanisms by which HSP70 functions as a target of CLC-conjugated drug delivery. In addition, HSP70 produced by tumor cells may undergo differential glycosylation, so kinetic studies tailored to each type of cancer may be required for more specific and accurate assessment of the binding affinity of HSP70 to CLC. The performance of a mutational study to identify putative binding site of CLC 57 is also required in the future to characterize thoroughly the interaction between CLC and HSP70.
Our data suggest that CLC targeted the primary tumor and metastatic lung lesions of 4T1 breast cancer in mice with high efficacy. While there was an increase in CLCs in the peripheral organs, in particular, the kidney; however, this retention faded after 3 days. Nonetheless, the tumor uptake of exogenously administered CLCs remains relatively stable over time.

| Preparation of CLC
Human CLC was expressed and optimized for an E. The purity of CLC was >85%, as estimated by a Coomassie bluestained SDS-PAGE gel. The concentration of CLC was determined by Bradford protein assay, using BSA as a standard. Its molecular weight was 28.1 kDa, and its isoelectric point was 4.37.

| Virtual screening of binding candidates to CLC
First, the 3D structure of CLC was established, according to its amino acid sequence. Then, screening was performed in silico to find interaction partners in the PDB database. GOR IV was used for secondary structure analysis of the protein. The 3D structure of the protein was generated by Homology Modeling Program, developed by Profacgen.
In total, 1590 human protein PDB entries were downloaded from www.rcsb.org. Three proteins with the highest binding energy were identified through AutoDock Vina: ADP-ribosylation factor 3, HSP70, and serine/threonine-protein phosphatase PP1-beta catalytic subunit (Table S1). Among these, HSP70 has commonly been reported as a cancer marker.

| Pharmacokinetics of CLC-PTX*
BALB/c mice (female; 7-8 weeks) were iv injected with PTX* or CLC-PTX* at an equivalent PTX* dose of 0.5 mg/kg. Sera from three mice were collected at 6 h and 2 days following drug administration.
The serum samples were stored at À20 C until analysis. The PTX plasma concentration was analyzed with the aid of the iBOX microscope.

| Therapeutic studies in tumor-bearing mice
BALB/c mice (female; 7-8 weeks) were implanted subcutaneously with 1.0 Â 10 5 4T1 cells in the left fourth mammary gland. C57BL/6 mice (female; 7-8 weeks) were inoculated subcutaneously with 1.0 Â 10 5 B16, LLC1, or Pan02 cells in the right rear flanks. When the tumor size reached~100 mm 3 , the mice were randomly divided into three groups (n = 6). All groups received treatments iv; the first group was injected with PBS (control), the second group with free PTX (total dose of PTX = 32 μg/kg), and the final group with CLC-PTX (total dose of CLC-PTX = 1.06 mg/kg, PTX dose identical to free PTX group). The treatment schedule consisted of twice-per-week injections for 2 weeks. The tumor size and body weight of the mice were monitored during the treatment course. The length (l) and width (w) of the tumor was measured by a digital Vernier caliper, and tumor volume (V) was defined as V ¼ l Â w 2 =2. We evaluated the TGI rate of

| Immunofluorescence staining
Frozen OCT blocks of tumors and LNs were cut using a cryostat

| Statistical analysis
All statistical analysis was conducted using GraphPad Prism 7 software (GraphPad Software, Inc., CA). The expression level of each marker in the immunostaining experiments was determined by dividing each DAPI level. The sample size (n) in the experiments was as follows: six for tumor growth analysis, six for lung metastatic foci assay, three for the fluorescent biodistribution, and three for the tissue immunostaining study. The fluorescence in vitro assay was independently conducted three times for statistical analysis. All data are expressed as the mean ± SD from at least three independent samples or experiments. Differences between the two groups were analyzed by an unpaired Student's t-test. Comparisons between multiple groups were determined using one or two-way analysis of variance with Holm-Sidak's post hoc test. A p value <0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

| CONCLUSION
In summary, CLC has significant potential to replace the antibodies used currently as delivery agents in ADCs, as it circumvents the complicated process of antibody optimization 6 as well as the limited targeting efficacy 6,7 of immunogenic and large-sized antibodies. Therefore, we expect CLC-drug conjugates to open a new window for targeted, protein-based delivery of drugs in cancer, as it may target metastatic lesions to enhance the survival rates in these cancer patients.

ACKNOWLEDGMENTS
This work was supported in part by the National Institutes of Health